Antibody-based therapy is an effective and clinically established treatment of various cancers, including solid tumors. For example, HERCEPTIN® has been used successfully to treat breast cancer and RITUXAN® is effective in B-cell related cancer types. Central to the development of a novel successful antibody-based therapy is the isolation of antibodies against cell-surface proteins found to be preferentially expressed on target cells (e.g. cancer cells, immune cells etc) that are able to functionally modify the activity of the corresponding receptor.
Antibody blockade of immune checkpoint molecules for immune cell activation and thus for immunotherapy of cancer is a clinically validated approach. In 2011 the CTLA-4 blocking antibody Ipilimumab has been approved by the FDA for the 2nd line therapy of metastatic melanoma (Yervoy). Another example is the blockade of the PD-1/PD-L1 axis for which several drugs are either approved or currently under clinical development and for which impressive clinical responses have been reported in melanoma, RCC and lung cancer (Henick et al., Expert Opin Ther Targets. 2014 December; 18(12):1407-20)).
Proteins of the Carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family belong to the immunoglobulin (Ig) supergene family and generally exhibit a variable (V)-like domain identified as the N domain. The N domain is followed by either none or up to six constant C2-like Ig domains (termed A or B). These extracellular domains are required for CEACAM functionality as homo- and heterophilic intercellular adhesion molecules (Obrinck, Curr Opin Cell Biol. 1997 October; 9(5):616-26) or as human and rodent pathogen receptors (Kuespert et al., Curr Opin Cell Biol. 2006 October; 18(5):565-71; Voges et al., PLoS One. 2012;7(6):e39908). CEACAM receptors associate as dimers or oligomers and multiple associations with other partners at the membrane and consequently modulate important functions. In addition to their expression in human tissues, the CEACAM gene family is highly conserved in 27 other mammalian species and is best described in mouse, rat, cattle, dog, platypus and opossum (Kammerer and Zimmermann, BMC Biol. 2010 Feb. 4; 8:12). The best characterized biological function of CEACAMs is the support of cell-cell adhesion through their homo- and heterophilic interactions, including a role in the differentiation and formation of a three-dimensional tissue structure, angiogenesis, apoptosis, tumor suppression, and metastasis. (Kuespert et al., Curr Opin Cell Biol. 2006 October; 18(5):565-71). More details on the family members are described in other reviews (Horst and Wagener, Handb Exp Pharmacol. 2004;(165): 283-341; Gray-Owen and Blumberg, Nat Rev Immunol. 2006 June; 6(6):433-46).
CEACAM6 (Carcinoembryonic antigen-related cell adhesion molecule 6, CD66c, Non-specific crossreacting antigen, NCA, NCA-50/90) is a glycosylphosphatidylinositol (GPI)-linked cell surface protein with one N-domain and 2 C2-like domains which mediate a number of possible cis or trans directed interactions of CEACAM proteins through their extracellular domains with a variety of membrane receptors, a few of which have been identified. (Beauchemin and Arabzadeh, Cancer Metastasis Rev. 2013 December; 32(3-4):643-71).
CEACAM6 is expressed in a variety of epithelia of normal human tissue such as colon (Blumenthal et al., BMC Cancer, 2007, Jan. 3; 7:2.), lung (Kolla et al., Am J Physiol Lung Cell Mol Physiol 296: L1019-L1030) and granulocytes (Kuroki et al., Biochem Biophys Res Commun. 1992 Jan. 31; 182(2):501-6). In the granulocytic lineage CEACAM6 was expressed at all stages of granulocytic maturation except for the early lineage-committed precursor cell (Strickland et al., J Pathol. 2009 July; 218(3):380-90); Schölzel et al., American Journal of Pathology, 156 (2), 595-605). CEACAM6 is not expressed in rodents. (Beauchemin et al., Exp Cell Res. 1999 Nov. 1; 252(2):243-9).
CEACAM6 expression has been described for several cancers. In colon cancer CEACAM6 is upregulated in 55% of the cases and an independent prognostic factor allowing subdivision of patients into low and high-risk groups (Jantscheff et al., J Clin Oncol. 2003 Oct. 1; 21(19):3638-46). In pancreatic adenocarcinoma 92% (n=82) of analyzed specimens were found to be positive while CEACAM6 expression was more prevalent in high-grade than in low grade PanIN lesions (Duxbury et al., Ann Surg. 2005 March; 241(3):491-6). This was confirmed in another study where >90% of invasive pancreatic adenocarcinomas (110 of 115 tested) showed a robust (over-) expression of CEACAM6 (Strickland et al., J Pathol. 2009 July; 218(3):380-90). In addition, Blumenthal et al. reported CEACAM6 expression in breast tumors, in pancreatic tumors, ovarian adenocarcinomas, lung adenocarcinoma, lymph node metastases and metastases from breast, colon and lung tumors. (Blumenthal et al., BMC Cancer. 2007 Jan. 3; 7:2).
CEACAM6 expression in breast cancer was also reported by others (Maraqa et al., Clin Cancer Res. 2008 Jan. 15; 14(2):405-11; Poola et al., Clin Cancer Res. 2006 Aug. 1;.12(15):4773-83; Balk-Moller et al., Am J Pathol. 2014 April; 184(4):1198-208); Tsang et al., Breast Cancer Res Treat. 2013 November; 142(2):311-22). In addition CEACAM6 expression has been reported in multiple myeloma (Witzens-Harig et al., Blood 2013 May 30; 121(22):4493-503), gastric cancer (Deng et al., Genet Mol Res. 2014 Sep. 26; 13(3):7686-97) and head and neck cancer (Cameron et al., Mol Cancer. 2012 Sep. 28; 11:74).
Experimental evidence supports a role for CEACAM6 as important regulator of metastasis. Kim et al. have shown that attenuating CEACAM6 expression in LoVo cells using a CEACAM6-specific siRNA or increasing its expression in HCT116 cells, respectively, impeded or augmented invasion through the extracellular matrix (Kim et al., Clin Chim Acta. 2013 Jan. 16; 415:12-9). Suppression of CEACAM6 expression leads to elevated E-cadherin promoter activity. Blumenthal et al. showed that CEACAM5 and CEACAM6 contributed to CRC metastatic dissemination which could be blocked by monoclonal antibodies in vivo. (Blumenthal et al., BMC Cancer. 2007 Jan. 3; 7:2). Also it has been shown that CEACAM6 is expressed in CD133-positive cells in colon cancer samples able to form stem cell-enriched colon spheres for which proliferation, clonogenic potential, as well as in vivo tumorigenic potential were significantly hampered upon its silencing (Gemei et al., Cancer. 2013 Feb. 15; 119(4):729-38). In breast cancer it was shown that tamoxifen resistant samples are CEACAM6 overexpressing and CEACAM6 was a significant predictor of recurrence of the disease (Maraqa et al., Clin Cancer Res. 2008 Jan. 15; 14(2):405-11). siRNA mediated CEACAM6 silencing in a MMU1-tamoxifen-resistant MCF7 cell derivative reversed endocrine resistance, anchorage independence of these cells and invasive properties (Lewis-Wambi et al., Eur J Cancer. 2008 August; 44(12):1770-9). In lung adenocarcinoma CEACAM6 expression was significantly associated with adverse clinical outcome (Kobayashi et al., Br J Cancer. 2012 Nov. 6; 107(10):1745-53). In pancreatic cancer CEACAM6 silencing with siRNA reversed the acquired anoikis resistance of Mia(AR) pancreatic tumor cells. Overexpression of CEACAM6 in Capan2 pancreatic cancer cells augmented gemcitabine resistance whereas siRNA-mediated suppression of CEACAM6 expression in BxPC3 cells chemosensitized them to the drug by modulating AKT activity in an Src dependent manner (Duxbury et al., Cancer Res. 2004 Jun. 1; 64(11):3987-93). These effects corresponded to increased invasiveness of high CEACAM6 expressing cells exhibiting c-src activity and matrix metalloproteinase (MMP9) expression (Duxbury et al., Br J Cancer. 2004 Oct. 4; 91(7):1384-90).
T-cell responses against tumor-associated antigens have been described in many tumors (Beckhove et al., J Clin Invest. 2004 July; 114(1):67-76; Choi et al., Blood. 2005 Mar. 1; 105(5):2132-4; Sommerfeldt et al., Cancer Res. 2006 Aug. 15; 66(16):8258-65; Schmitz-Winnenthal et al., Cancer Res. 2005 Nov. 1; 65(21):10079-87.; Jäger et al., Proc Natl Acad Sci USA. 2000 Apr. 25; 97(9):4760-5; Romero et al., Adv Immunol. 2006; 92:187-224) and often cause an accumulation of tumor specific memory T cells in lymphoid organs or in the blood (Choi et al., Blood. 2005 Mar. 1; 105(5):2132-4; Feuerer et al., Nat Med. 2001 April; 7(4):452-8; Letsch et al., Cancer Res. 2003 Sep. 1; 63(17):5582-6). However, the capacity of T cells to react against autologous tumor cells is generally low (Horna and Sotomayor, Curr Cancer Drug Targets. 2007 February; 7(1):41-53); Yang and Carbone, Adv Cancer Res. 2004; 92:13-27). Many tumors have the capacity to block effector functions of T cells which contributes to the limited activity of tumor immunotherapy. T-cell unresponsiveness against tumor cells has been demonstrated for a broad variety of cancers (Pardoll, Nat Immunol. 2012 December; 13(12):1129-32).
CEACAM6 also contributes to the regulation of CD8+ T cell response. Recently, Witzens-Harig et al. demonstrated in multiple myeloma expressing several CEACAM family members that treatment with anti-CEACAM6 mAbs or siRNA silencing CEACAM6 reinstated T cell reactivity against malignant plasma cells indicating a role for CEACAM6 in CD8+ T cell response regulation (Witzens-Harig et al., Blood 2013 May 30; 121(22):4493-503). So far, a receptor for CEACAM6 on T cells has not been identified. However, co-culture of CEACAM6 positive myeloma cells with T cells resulted in the modulation of T cell signaling events including an activation of SHP phosphatases by CEACAM6 ligation (Lin and Weiss, J Cell Sci. 2001 January; 114(Pt 2):243-4; Latour et al., Mol Cell Biol. 1997 August; 17(8):4434-41; Wen et al., J Immunol. 2010 Dec. 1; 185(11):6413-9). CEACAM6 has no intrinsic signaling capacity, and its inhibitory capacity is presumably mediated by binding to receptors on the T cell surface. Such a receptor can be for example CEACAM1 for which mechanism for the modulation of innate and adaptive immune responses have been described. CEACAM1 (CD66a) possesses a cytoplasmic tail containing an immunoreceptor tyrosine-based inhibitory (ITIM) motif. CEACAM1 is stored in intracellular vesicles and upon T cell activation is rapidly (24 h to 72 h) externalized and expressed on the T cell surfacewhere it mediates the blockade of T-cell effector functions after homo- or heterophilic binding to ligands expressed on target cells (Gray-Owen and Blumberg, Nat Rev Immunol. 2006 June; 6(6):433-46). The nature of this binding is unknown and could be either homo- or heterophilic binding to other CEACAMs or binding to other components of the extracellular matrix, growth factor receptors, integrins, or cadherins. Homophilic interactions have been reported between CEACAM1 and CEACAM1 (Ortenberg et al., Mol Cancer Ther. 2012 June; 11(6):1300-10). Heterophilic CEACAM interactions have been described for example between CEACAM1 and CEACAM5, and CEACAM6 and CEACAM8 (Cavallaro and Christofori, Nat Rev Cancer. 2004 February; 4(2):118-32).
As described above CEACAM6 is a very attractive target for therapeutic intervention in cancer immunotherapy. As noted, CEACAM6 is a member of a family of highly homologous proteins. An antibody suitable for human therapy, which is relieving immunosuppression of CEACAM6, must therefore be able to distinguish between CEACAM6 and other paralogous proteins like CEACAM1, CEACAM3, CEACAM5, which each display different functions and tissue distributions, to restrict its mode of action and localization to CEACAM6 and to avoid unwanted adverse side effects.
As CEACAM6 is not only expressed on tumor cells but also on normal tissues (especially granulocytes but also epithelial cells of e.g. lung and gastrointestinal cells—Chan and Stanners, Mol Ther. 2004 June; 9(6):775-85; Strickland et al., J Pathol. 2009 July; 218(3):380-90), it is absolutely crucial to be able to predict the adverse side effect profile of the therapeutic antibody. This is all the more important, since the anticipated mode of action will be inhibition of immunosuppression, i.e. an immunoactivation, which can result in serious hazards (incident of CD28 superagonist TGN1412 trial; Suntharalingam et al., N Engl J Med. 2006 Sep. 7; 355(10):1018-28). So indirect effects on the immune system on top of direct effects on granulocytes need to be carefully assessed. To enable the development of a human therapeutic antibody and a predictive pre-clinical tolerability testing, it is mandatory for the antibody to exhibit relevant cross-reactivity to a toxicology relevant species, in case of CEACAM6 to non-human primates, preferentially Macaca fascicularis (cynomolgus).
As a prerequisite, a therapeutic antibody needs to bind with high affinity to human CEACAM6 on cells, to bind selectively to CEACAM6 (without binding to any paralogs), to be cross-reactive to monkey CEACAM6 within one order of magnitude of monovalent KD (to safely reflect binding on normal tissues in the toxicology monkey model even at low surface densities under non-avidity based binding conditions), to bind to a similar epitope as on human CEACAM6, to be able to relieve CEACAM6-mediated immunosuppression, to be non-immunogenic in human therapy (i.e. a human or humanized antibody), and to be stable enough to allow for clinical development, formulation and storage over extended periods of time as a pharmaceutical. The latter is important as it has been noted earlier that physical degradation (especially aggregation) may enhance immune response to a therapeutic protein (Hermeling et al., Pharm Res. 2004 June; 21(6):897-903) and aggregation is closely connected to unfolding of IgG and its thermal stability (Vermeer and Norde, Biophys J. 2000 January; 78(1):394-404).
Several anti-CEACAM6 antibodies exist. Most of them are non-human reagent antibodies, many of them are polyclonal. The specificity and selectivity to human CEACAM6 as well as cross-reactivity to monkey CEACAM6 is in most of the cases not disclosed or known.
Therapeutic antibodies directed against CEACAM6 are also known in the art. Some are not selective to human CEACAM6 (e.g. MN-3 from Immunomedics, Neo201/h16C3 from Neogenix; both binding in addition to human CEACAM5). A single domain antibody 2A3 and its fusion variants (WO2012040824 and Niu et al., J Control Release. 2012 Jul. 10; 161(1):18-24) are not characterized with respect to selectivity and cross-reactivity to monkey CEACAM6.
Selective anti-CEACAM6 antibodies apparently cross-reactive to monkey CEACAM6 are not disclosed (Strickland et al., J Pathol. 2009 July; 218(3):380-90).
The murine antibody 9A6 (Genovac/Aldevron) is the only antibody described to be able to modulate the immunosuppressive activity of CEACAM6 (Witzens-Harig et al., Blood 2013 May 30; 121(22):4493-503). 9A6 inhibits the immunosuppressive activity of CEACAM6, leading to enhanced cytokine secretion by T cells in vitro and anti-tumor efficacy in vivo (Khandelwal et al., Poster Abstract 61, Meeting Abstract from 22nd Annual International Cancer Immunotherapy Symposium Oct. 6-8, 2014, New York City, USA). Although its selectivity appears appropriate, it was previously not characterized with regards to its cross-reactivity to monkey CEACAM6. In addition, its murine nature precludes a direct therapeutic application in humans.
As shown in the examples, the antibody 9A6 binds to recombinant human CEACAM6 but no binding to recombinant Macaca mulatta or Macaca fascicularis CEACAM6 was detected. For comparison, Neo201-hIgG1 was also tested. This antibody displayed high affinity binding to both human and monkey CEACAM6. But Neo201 binds to human CEACAM5 and CEACAM6 and is therefore not specific for CEACAM6.
In conclusion there is high need for a therapeutic monoclonal antibody that comprises the following features:                i. The antibody is a high affinity binder of human CEACAM6.        ii. The antibody is selective to CEACAM6, not binding to any paralogs, especially CEACAM1, CEACAM3, and CEACAM5.        iii. The antibody is cross-reactive to monkey CEACAM6 within one order of magnitude of monovalent KD.        iv. The antibody is non-immunogenic in human therapy, i.e. it is a human or humanized antibody.        v. The antibody is able to relieve CEACAM6-mediated immunosuppression.        
Such an antibody does not exist in the prior art. 9A6 binding to N-terminal domain 1 of human CEACAM6 is the only known anti-CEACAM6 antibody that is able to relieve CEACAM6-mediated immunosuppression, yet lacks cross-reactivity to monkey CEACAM6 apart from being a mouse antibody. Neo201 binds to a different domain outside of N-terminal domain 1 of CEACAM6. Therapeutic efficacy of Neo201-hIgG1 has been published to be based on ADCC (Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr. 2-6; Orlando, Fla. Philadelphia (PA): AACR: Du et al., Cancer Res Apr. 15, 2011; 71(8 Supplement): 4582).
The inventors assumed that relief of CEACAM6-mediated immunosuppression is connected to binding to N-terminal domain 1. But generation of antibodies binding to N-terminal domain 1 of CEACAM6 results in a challenging selectivity problem.
The sequence alignment in FIG. 1 shows a very high degree of similarity of protein sequences of human CEACAM6 and human CEACAM3, human CEACAM5 and human CEACAM1 throughout the entire extracellular region. The target region (domain 1 of human CEACAM6) is especially similar to other CEACAMs, which is also reflected in Table 7. The paralogs of human CEACAM6 (e.g. CEACAM1, CEACAM3, and CEACAM5) are much more similar to human CEACAM6 than the cynomolgus ortholog. In fact, there are only 2 positions in the N-terminal region in the primary sequence that are identical in human and cynomolgus CEACAM6 but different from amino acids in the other human paralogs (marked in FIG. 1 with asterisks).
Unexpectedly the inventors were able to find a method to generate antibodies comprising all of the desired selectivity and functional features.